Many existing anti-cancer chemotherapeutics are non-specific, in that they typically damage or kill normal cells as well as malignant cells. Research in oncology is increasingly focused on targeted therapies, in which a therapeutic compound interacts with a specific molecule to interfere with a particular molecular pathway. Tumors in different individuals, even when found at the same anatomic location, can differ in their molecular signalling pathways. Accordingly, it is important to know which molecules and pathways are targeted by a therapeutic compound, so that the treatment can be provided to the appropriate patients. Determining which molecules and pathways are affected by a therapeutic compound also provides diagnostic techniques to identify those patients suitable for treatment with that therapeutic.
ErbB Receptors
The ErbB family of type I receptor tyrosine kinases includes ErbB1 (also known as the epidermal growth factor receptor (EGFR or HER1)), ErbB2 (also known as Her2), ErbB3, and ErbB4. These receptor tyrosine kinases are widely expressed in epithelial, mesenchymal, and neuronal tissues where they play a role in regulating cell proliferation, survival, and differentiation (Sibilia and Wagner, Science, 269: 234 (1995); Threadgill et al., Science, 269: 230 (1995)). Increased expression of wild-type ErbB2 or EGFR, or expression of constitutively activated receptor mutants, transforms cells in vitro (Di Fiore et al., 1987; DiMarco et al., Oncogene, 4: 831 (1989); Hudziak et al., Proc. Natl. Acad. Sci. USA., 84: 7159 (1987); Qian et al., Oncogene, 10: 211 (1995)). Increased expression of ErbB2 or EGFR has been correlated with a poorer clinical outcome in some breast cancers and a variety of other malignancies (Slamon et al., Science, 235: 177 (1987); Slamon et al., Science, 244: 707 (1989); Bacus et al, Am. J. Clin. Path., 102: S13 (1994)).
A family of peptide ligands binds to and activates ErbB receptor signaling, and includes epidermal growth factor (EGF) and transforming growth factor α (TGF-α), each of which binds to EGFR (Reise and Stem, Bioessays, 20: 41 (1998); Salomon et al., Crit. Rev. Oncol. Hematol., 19: 183 (1995)). Ligand-receptor interactions are selective in that epidermal growth factor (EGF) and transforming growth factor alpha (TGFα) bind EGFR while heregulin binds ErbB3 and ErbB4. Ligand binding induces ErbB receptor phosphorylation (activation) with subsequent formation of homo- and heterodimers. ErbB2 is the preferred heterodimeric partner for EGFR, ErbB3, and ErbB4 (Graus-Porta et al., EMBO J., 16: 1647 (1997); Tzahar et al., Mol. Cell. Biol., 16: 5276 (1996)). A number of soluble ligands have been identified for EGFR, ErbB3, and ErbB4, but none have been identified for ErbB2, which seems to be transactivated following heterodimerization (Ullrich and Schlessinger, Cell, 61: 203 (1990); Wada et al., Cell, 61: 1339 (1990); Karunagaran et al., EMBO J., 15: 254 (1996); Stem and Kamps, EMBO J., 7: 995 (1988)).
Truncated ErbB2
The ErbB2 gene encodes a Mr 185,000 member of the ErbB family. The full-length ErbB2 receptor (p185ErbB2) undergoes proteolytic cleavage releasing its extracellular domain (ECD), which can be detected in cell culture medium and in patient's sera. (Lin and Clinton, Oncogene 6: 639 (1991); Zabrecky et al., J. Biol. Chem. 266: 1716 (1991); Pupa et al., Oncogene 8: 2917 (1993)). Cleavage of ErbB2 appears to be mediated by a member of the matrix metalloprotease (MMP) family (Codony-Servat et al., Cancer Res. 59: 1196 (1999)). The truncated ErbB2 receptor (p95ErbB2) that remains after proteolytic cleavage exhibits increased autokinase activity and transforming efficiency compared with the full-length receptor, implicating the ErbB2 ECD as a negative regulator of ErbB2 kinase and oncogenic activity. (Di Fiore et al., Science 237: 178 (1987); Bargmann and Weinberg, EMBO J 7: 2043 (1988); Segatto et al., Mol. Cell. Biol. 8: 5570 (1988)).
Expression of the p95ErbB2 truncated ErbB2 receptors in breast cancer cells has been correlated with positive lymph node metastasis in ErbB2 overexpressing tumors. (Christianson et al., Cancer Res. 58: 5123 (1998); Molina et al., Clin. Cancer Res. 8: 347 (2002)). Elevated serum levels of ErbB2 ECD in women with breast cancer has also been correlated with a poorer response to therapy. (Brandt-Rauf, Mutat. Research 333: 203 (1995); Kandl et al., Br. J. Cancer 70: 739 (1994); Yamauchi et al., J. Clin. Oncol. 15: 2518 (1996); Colomer et al., Clin. Cancer Research 6: 2356 (2000)).
Therapeutics and ErbB2
Trastuzumab (Herceptin™), a humanized anti-ErbB2 monoclonal antibody has been approved for the treatment of breast cancers that either overexpress ErbB2, or that demonstrate ErbB2 gene amplification (Cobleigh et al, J. Clin. Oncol., 17: 2639 (1999)). Trastuzumab binds to the extracellular domain of the ErbB2 receptor, and has been reported to exert its antitumor effects through several mechanisms. See e.g., Sliwkowski et al., Semin. Oncol. 26(Suppl 12): 60 (1999). In ErbB2 over-expressing cells, trastuzumab has been reported to down-regulate ErbB2 expression (Sarup et al., Growth Reg 1: 72 (1991); Lane et al., Mol. Cell Biol. 20: 3210 (2000)). In animal models, trastuzumab has been reported to induce antibody-dependent cell-mediated cytotoxicity against ErbB2 expressing tumor cells (Clynes et al., Nat. Med. 6: 443 (2000)). Molina et al., Cancer Research 61: 4744 (2001) report that trastuzumab reduced ECD shedding from two breast adenocarcinoma cell lines, whereas another antibody (2C4) directed against the ErbB2 ectodomain did not.
Combination therapy with trastuzumab and chemotherapy has been associated with a longer time to disease progression in breast cancer, a longer duration of response, and longer survival, compared to chemotherapy alone. Slamon et al., NEJM 344: 783 (2001). However, resistance to trastuzumab frequently occurs within the first year of treatment. See e.g., Baselga et al. Eur J Cancer, 37 Suppl 1: 18 (2001). Strategies to reduce or prevent resistance to trastuzumab are needed; one such proposed strategy is to target insulin-like growth factor I receptor (IGF-IR) signaling to delay development of trastuzumab resistance. See, e.g., Lu et al., J Natl Cancer Inst. 2001 Dec. 19; 93(24): 1852-7; Camirand et al., Med Sci Monit. 8: BR521 (2002).
Because heterodimers of ErbB2 and EGFR can elicit potent mitogenic signals, interrupting both ErbB2 and EGFR simultaneously is a potential therapeutic strategy (Earp et al., Breast Cancer Res. Treat., 35: 115 (1995)). Small molecule, dual EGFR-ErbB2 tyrosine kinase inhibitors have been identified and their pre-clinical anti-tumor activities reported (Fry et al., Proc. Natl. Acad. Sci. USA., 95: 12022 (1998); Cockerill et al., Bioorganic Med. Chem. Letts., 11: 1401 (2001); Rusnak et al., Cancer Res., 61: 7196 (2001); Rusnak et al., Mol. Cancer Therap., 1: 85 (2001)).
GW572016 (lapatinib) is a potent reversible, dual inhibitor of the tyrosine kinase domains of both EGFR and ErbB2, with IC50 values against purified EGFR and ErbB2 of 10.2 and 9.8 nM, respectively (Rusnak et al., Mol. Cancer Therap., 1: 85 (2001)). Recent reports have demonstrated that GW572016 inhibits EGFR and ErbB2 autophosphorylation in tumor cell lines that overexpress these receptors (Rusnak et al., Mol. Cancer Therap., 1: 85 (2001)), an effect that was primarily associated with tumor cell growth arrest. The chemical name of GW572016 is N-{3-chloro-4-[(3-fluorobenzyl)oxy]phenyl}-6-[5-({[2-methylsulfonyl)ethyl]amino}methyl)-2-furyl]-4-quinazolinamine (WO 99 35146, Carter et al.); a ditosylate form is disclosed in WO 02 02552 (McClure et al); methods of treating cancer are disclosed in WO 02/056912, and PCT/US03/10747.